Transcriptional polarity in rRNA operons of Escherichia coli nusA and nusB mutant strains - PubMed (original) (raw)

Transcriptional polarity in rRNA operons of Escherichia coli nusA and nusB mutant strains

Selwyn Quan et al. J Bacteriol. 2005 Mar.

Abstract

Synthesis of ribosomes in Escherichia coli requires an antitermination system that modifies RNA polymerase to achieve efficient transcription of the genes specifying 16S, 23S, and 5S rRNA. This modification requires nucleotide signals in the RNA and specific transcription factors, such as NusA and NusB. Transcription of rrn operons in strains lacking the ability to produce either NusA or NusB was examined by electron microscopy. The distribution and numbers of RNA polymerase molecules on rrn operons were determined for each mutant. Compared to the wild type, the 16S gene in the nusB mutant strain had an equivalent number of RNA polymerase molecules, but the number of RNA polymerase molecules was reduced 1.4-fold for the nusA mutant. For both mutant strains, there were twofold-fewer RNA polymerase molecules on the 23S RNA gene than for the wild type. Overall, the mutant strains each had 1.6-fold-fewer RNA polymerase molecules on their rrn operons than did the wild type. To determine if decreased transcription of the 23S gene observed by electron microscopy also affected the 30S/50S ribosomal subunit ratio, ribosome profiles were examined by sucrose gradient analysis. The 30S/50S ratio increased 2.5- to 3-fold for the nus mutant strains over that for wild-type cells. Thus, strains carrying either a nusA mutation or a nusB mutation have defects in transcription of 23S rRNA.

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Figures

FIG. 1.

Schematic diagram of the rrnB operon showing the regulatory BoxBAC region and its position relative to the 16S gene in the leader and the 23S gene in the spacer region.

FIG. 2.

Electron micrographs of rrn operons. A. Chromosomal rrn operon from a wild-type strain. B. rrn operon from a nusA::cat rhoE134D mutant strain. C. rrn operon from a nusB::IS_10_ mutant strain. rrn operons are all 5.5 kb in length.

FIG. 3.

Determination of interpolymerase distance. The distance from polymerase center to polymerase center of all adjacent polymerases was measured. A. Interpolymerase spacing in the wild-type strain (n = 217 for 16S and n = 356 for 23S from 11 operons). B. Interpolymerase spacing in the nusB::IS_10_ mutant strain (n = 459 for 16S and n = 444 for 23S from 27 operons). Closed columns indicate polymerase molecules on the 16S genetic region, and open columns represent polymerase molecules on the 23S genetic region.

FIG. 4.

Distribution of RNA polymerase molecules on rrn operons normalized to total polymerase numbers. A. Measurements of RNA polymerase numbers on rrn operons for the wild-type strain (n = 11). B. Measurements of RNA polymerase numbers for the nusB::IS_10_ mutant strain (n = 27).

FIG. 5.

Sucrose gradient analysis of ribosomes prepared from the following: A. wild-type strain, MC4100; B. nusB::IS_10_ mutant strain; and C. nusA::cat rhoE134D mutant strain.

FIG. 6.

Diminished transcription of the 23S genetic region in a nusA or nusB mutant strain due to premature termination.

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Transcriptional polarity in rRNA operons of Escherichia coli nusA and nusB mutant strains - PubMed (original) (raw)